The archaebacteria are a recently discovered group of microorganisms that grow optimally at temperatures above 80.degree. C. Some 20 species of these extremely thermophilic bacteria-like organisms have been isolated, mainly from shallow submarine and deep sea geothermal environments. Most of the archaebacteria are strict anaerobes and depend on the reduction of elemental sulfur for growth.
The archaebacteria include a group of "hyperthermophiles" that grow optimally around 100.degree. C. These are presently represented by three distinct genera, Pyrodictium, Pyrococcus, and Pyrobaculum. Phyodictium brockii (T.sub.opt 105.degree. C.) is an obligate autotroph which obtains energy be reducing S.sup.0 to H.sub.2 S with H.sub.2, while Pyrobaculum islandicum (T.sub.opt 100.degree. C.) is a facultative heterotroph that uses either organic substrates or H.sub.2 to reduce S.sup.0. In contrast, Pyrococcus furiosus (T.sub.opt 100.degree. C.) grows by a fermentative-type metabolism rather than by S.sup.0 respiration. It is a strict heterotroph that utilizes both simple and complex carbohydrates where only H.sub.2 and CO.sub.2 are the detectable products. The organism reduces elemental sulfur to H.sub.2 S apparently as a form of detoxification since H.sub.2 inhibits growth.
The discovery of microorganisms growing optimally around 100.degree. C. has generated considerable interest in both academic and industrial communities. Both the organisms and their enzymes have the potential to bridge the gap between biochemical catalysis and many industrial chemical conversions. However, knowledge of the metabolism of the hyperthermophilic microorganisms is presently very limited.
The polymerase chain reaction (PCR) is a powerful method for the rapid and exponential amplification of target nucleic acid sequences. PCR has facilitated the development of gene characterization and molecular cloning technologies including the direct sequencing of PCR amplified DNA, the determination of allelic variation, and the detection of infectious and genetic disease disorders. PCR is performed by repeated cycles of heat denaturation of a DNA template containing the target sequence, annealing of opposing primers to the complementary DNA strands, and extension of the annealed primers with a DNA polymerase. Multiple PCR cycles result in the exponential amplification of the nucleotide sequence delineated by the flanking amplification primers.
An important modification of the original PCR technique was the substitution of Thermus aquaticus (Taq) DNA polymerase in place of the Klenow fragment of E. coli DNA pol I (Saiki, et al. Science, 230:1350-1354 (1988)). The incorporation of a thermostable DNA polymerase into the PCR protocol obviates the need for repeated enzyme additions and permits elevated annealing and primer extension temperatures which enhance the specificity of primer:template associations. Thermostability in a polymerase thus serves to increase the specificity and simplicity of PCR.
Recently, a thermostable DNA polymerase was isolated from P. furiosus (Pfu), referred to as Pfu DNA polymerase. Lundberg et al., Gene, 108:1-6 (1991). Pfu DNA polymerase exhibits a higher temperature optimum than Taq DNA polymerase, thereby making it an important thermostable DNA polymerase.
Linear amplification sequencing, also known as cycle sequencing, is rapidly becoming the method of choice for sequencing difficult templates. Murray, Nuc. Acids Res., 17:8889 (1989); and Craxton, Methods: A Companion to Methods in Enzymology, 3:20-26 (1991). PCR products, direct colonies and phage plaques can be sequenced using the cycle sequencing procedure. to date, Taq DNA polymerase has been almost exclusively used for cycle sequencing reactions.
However, there are several inherent disadvantages to using Taq DNA polymerase in cycle sequencing. Taq DNA polymerase incorporates thiolated nucleotides such as .sup.35 S-dATP very inefficiently because the enzyme discriminates highly against ddNTP analogs. .sup.35 S-dATP is the most widely used label in DNA sequencing today, at least because it is considerably safer to use than the other common labels such as .sup.32 p- or .sup.33 P-labelled dNTP's. In addition, Taq DNA polymerase loses considerable activity when used in cycling reactions at temperatures required for template denaturation, such as cycle sequencing reactions.
DNA polymerase from Thermococcus litoralis has recently been described ("Vent" polymerase) which has significant thermostability. However, cycle sequencing with Vent DNA polymerase exhibits an additional problem referred to as "stuttering" which arises from the editing function present in the 3' to 5' exonuclease activity present in the enzyme. Stuttering leads to excessive bands in the sequencing reaction and produces unreadable sequencing gels. The thermolabile DNA polymerase from E. coli has been characterized toy contain protein domains responsible for the 3' to 5' exonuclease activity as described by Morrison et al., Proc. Natl. Acad. Sci. USA, 88:9473-9477 (1991). However, similar domains have not been identified in a thermostable DNA polymerase. Thus there is a need for thermostable DNA polymerases other than Vent DNa polymerase with less 3' to 5' HP LaserJet Series IIHPLASEII.PRS Vent and CircumVent are available from New England Biolabs (NEB) and have been described by Sears et al., Biotechniques, 13:626-633 (1992). However, both enzymes exhibit thermo-instability upon prolonged exposure to temperatures of about 95.degree. C. that are often used in cycle sequencing.
There continues to be a need for a highly thermostable DNA polymerase useful in cycle sequencing which is deficient in 3'-5' exonuclease activity and which efficiently incorporates thiolated dNTP analogs in DNA polymerization reactions.